Method for producing artificial recombinant rotavirus

ABSTRACT

Provided is a method for producing an artificial recombinant virus of the family Reoviridae, the method comprising the steps of:
     (1) introducing a FAST protein expression vector and/or a capping enzyme expression vector into host cells;   (2) introducing a vector containing expression cassettes for individual RNA genome segments of a virus or introducing a set of single-stranded RNA transcripts from the expression cassettes into host cells; and   (3) culturing the host cells.   

     The method of the present invention allows more efficient production of an artificial recombinant virus of the family Reoviridae as compared with conventional methods and allows artificial recombinant rotavirus production without using a helper virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCTInternational Application Number PCT/JP2017/034783, filed on Sep. 26,2017, designating the United States of America and published in theJapanese language, which is an International Application of and claimsthe benefit of priority to Japanese Patent Application No. 2016-188881,filed on Sep. 27, 2016, and Japanese Patent Application No. 2017-068323,filed on Mar. 30, 2017. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52(e). Thename of the ASCII text file for the Sequence Listing isSeqList-IWAT007-003APC.txt, the date of creation of the ASCII text fileis Mar. 5, 2019, and the size of the ASCII text file is 75 KB.

TECHNICAL FIELD

The present invention relates to a method for producing an artificialrecombinant virus of the family Reoviridae, particularly to a method forproducing an artificial recombinant rotavirus.

BACKGROUND ART

Rotaviruses, members of the family Reoviridae, are known as a causativevirus of infant diarrhea. Infants aged from 6 months to 2 years are athigh risk of rotavirus infection and rotavirus disease development.Almost all children will have been infected with rotaviruses by the ageof five. Vaccines against rotaviruses are in practical use and theirpreventive efficacy has been proven in practice. In the meanwhile,next-generation rotavirus vaccines that are less expensive and havehighly preventive effect are under research and development.

Reverse genetics (RG) systems that allow artificial virus productionhave been established for a wide variety of RNA viruses and have greatlycontributed to the progress of virological basic research and appliedresearch such as viral vector development and vaccine vectordevelopment. However, the development of RG systems for Reoviridaeviruses, which have a 10 to 12 segmented double-stranded RNA (dsRNA)genome, lags behind that of RG systems for other RNA viruses due to thecomplexity of their segmented genome.

Various RG systems for Reoviridae viruses have been developed so far.For bluetongue virus and African horse sickness virus in the genusOrbivirus, RNA-based RG systems have been developed, and these systemsallow recombinant virus production based on the introduction of viralRNA into cells (Non Patent Literature 1 and 2). For Mammalianorthoreovirus in the genus Orthoreovirus, an entirely DNA-based RGsystem using cDNA has been developed (Non Patent Literature 3). Forrotaviruses in the genus Rotavirus, partially DNA-based RG systems usinga helper virus have been reported (Non Patent Literature 4 and 5).However, the helper virus-dependent RG systems have disadvantages inthat a potent means of separating the virus of interest from the helpervirus is required; that mutation can be introduced only into limitedtypes of segment genes (VP4 gene, NSP2 gene); and that productionefficiency is low. Under such circumstances, the development of completeRG systems that allow rotavirus production based on the introduction ofonly cDNA or RNA without using a helper virus is eagerly anticipated.

CITATION LIST Non Patent Literature

Non Patent Literature 1:

-   Boyce, M., Celma, C. C., and Roy, P., Development of reverse    genetics systems for bluetongue virus: recovery of infectious virus    from synthetic RNA transcripts, J Virol 82:8339-8348, 2008.    Non Patent Literature 2:-   Kaname Y, Celma C C, Kanai Y, Roy P., Recovery of African horse    sickness virus from synthetic RNA, J Gen Virol 94:2259-2265, 2013.    Non Patent Literature 3:-   Kobayashi, T, Antar, A A R, Boehme, K W, Danthi, P, Eby, E A,    Guglielmi, K M, Holm, G H, Johnson, E M, Maginnis, M S, Naik, S,    Skelton, W B, Wetzel, J D, Wilson, G J, Chappell, J D, and Dermody,    T S, A plasmid-based reverse genetics system for animal    double-stranded RNA viruses. Cell Host Microbe 1:147-157, 2007.    Non Patent Literature 4:-   Komoto, S, Sasaki, J, and Taniguchi, K, Reverse genetics system for    introduction of site-specific mutations into the double-stranded RNA    genome of infectious rotavirus. Proc Natl Acad Sci USA    103:4646-4651, 2006.    Non Patent Literature 5:-   Trask S D, Taraporewala Z E, Boehme T S, Dermody T S, Patton J T,    Dual selection mechanisms drive efficient single-gene reverse    genetics for rotavirus. Proc Natl Acad Sci USA 107:18652-18657 2010.

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a method for producingan artificial recombinant virus of the family Reoviridae using animproved reverse genetics system for Reoviridae viruses, which method ismore efficient in virus production as compared with conventional ones.Another object of the present invention is to provide a method forproducing an artificial recombinant rotavirus without using a helpervirus. A yet another object of the present invention is to provide anartificial recombinant rotavirus as a vaccine candidate, the artificialrecombinant rotavirus having a mutation introduced in a viral genomesegment.

Solution to Problem

The present invention includes the following to achieve theabove-mentioned objects.

[1] A method for producing an artificial recombinant virus of the familyReoviridae, the method comprising the steps of:

(1) introducing a FAST protein expression vector and/or a capping enzymeexpression vector into host cells;

(2) introducing a vector containing expression cassettes for individualRNA genome segments of a virus or introducing a set of single-strandedRNA transcripts from the expression cassettes into host cells; and

(3) culturing the host cells.

[2] The method according to the above [1], wherein the artificialrecombinant virus has a mutation introduced in at least one of the RNAgenome segments and/or a foreign gene inserted in at least one of theRNA genome segments.

[3] The method according to the above [1] or [2], wherein the FASTprotein is at least one kind selected from Nelson Bay reovirus p10,Avian reovirus p10, Broome reovirus p13, Reptilian reovirus p14, Baboonreovirus p15, grass carp reovirus p16 and Atlantic salmon reovirus p22.[4] The method according to any one of the above [1] to [3], wherein thecapping enzyme is a capping enzyme of a DNA or RNA virus whichreplicates in the cytoplasm of host cells.[4-1] The method according to any one of the above [1] to [3], whereinthe capping enzyme is a capping enzyme of a virus of the familyPoxviridae.[5] The method according to any one of the above [1] to [4], wherein theexpression cassette for an RNA genome segment comprises an RNApolymerase promoter, a DNA encoding the RNA genome segment and a DNAencoding a self-cleaving ribozyme.[6] The method according to the above [5], wherein the RNA polymerasepromoter is T7 promoter, and the host cells are recombinant T7 RNApolymerase-expressing cells.[7] The method according to the above [5] or [6], wherein the ribozymeis a hepatitis D virus ribozyme.[8] The method according to any one of the above [1] to [7], wherein thehost cells are co-cultured with highly virus-susceptible cells.[9] The method according to any one of the above [1] to [8], wherein theartificial recombinant virus of the family Reoviridae is an artificialrecombinant rotavirus.[10] The method according to the above [9], comprising overexpressing arotavirus NSP2 gene product and/or a rotavirus NSP5 gene product in thehost cells.[11] The method according to the above [9] or [10], wherein theartificial recombinant rotavirus expresses a foreign gene, and wherein avector containing an expression cassette for an RNA genome segmentencoding NSP1 which cassette has an insertion of the foreign gene in anNSP1 gene and a 100- to 1550-base deletion in the NSP1 gene is usedinstead of a vector containing an expression cassette for an RNA genomesegment encoding NSP1.[12] A method for promoting viral replication, comprising infecting hostcells expressing a FAST protein with a virus and culturing the hostcells.[13] The method according to the above [12], wherein the FAST protein isat least one kind selected from Nelson Bay reovirus p10, Avian reovirusp10, Broome reovirus p13, Reptilian reovirus p14, Baboon reovirus p15,grass carp reovirus p16 and Atlantic salmon reovirus p22.[14] An artificial recombinant rotavirus having a mutation resulting infunctional suppression of at least one selected from NSP1, NSP3 andNSP4.[15] An artificial recombinant rotavirus expressing a foreign gene.[16] An artificial recombinant reassortant rotavirus.[17] A vaccine comprising the artificial recombinant rotavirus accordingto any one of the above [14] to [16].[18] A method for producing an artificial recombinant rotavirus,comprising introducing a vector containing expression cassettes for 11individual RNA genome segments of a rotavirus or introducing a set of 11single-stranded RNA transcripts from the expression cassettes into hostcells expressing neither a FAST protein nor a capping enzyme, andculturing the host cells.[19] The method according to the above [18], comprising overexpressing arotavirus NSP2 gene product and/or a rotavirus NSP5 gene product in thehost cells, and culturing the host cells.[20] A method for producing an artificial recombinant virus of thefamily Reoviridae, the method comprising

introducing a vector containing expression cassettes for individual RNAgenome segments of a virus or introducing a set of single-stranded RNAtranscripts from the expression cassettes into host cells;

overexpressing, in the host cells, a gene product involved in theformation of viral inclusion bodies in infected cells; and

culturing the host cells.

Advantageous Effects of Invention

The present invention provides a method for producing an artificialrecombinant virus of the family Reoviridae using a reverse geneticssystem that allows more efficient artificial recombinant virusproduction as compared with conventional ones. Also provided is a methodfor producing an artificial recombinant rotavirus without using a helpervirus, which method has not been available so far. Also provided is anartificial recombinant rotavirus as a vaccine candidate, the artificialrecombinant rotavirus having a mutation introduced in a viral genomesegment.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 graphically illustrates the results of the enhancement effect onthe efficiency of artificial recombinant virus production using aMammalian orthoreovirus reverse genetics system by co-expression with aFAST protein and/or a capping enzyme in host cells.

FIG. 2 shows the mutation sites of plasmids having a marker mutation(s)used for artificial recombinant rotavirus production. NSP1wt (SEQ ID NO:34), NSP2 wt (SEQ ID NO: 35), NSP3 wt (SEQ ID NO: 36), NSP4 wt (SEQ IDNO: 37).

FIG. 3 shows the results confirming that the viruses produced using arotavirus reverse genetics system have a marker mutation(s).

FIG. 4 shows the structures of a wild-type NSP1 gene and a deletionmutant of the NSP1 gene.

FIG. 5 shows the results of SDS-PAGE of the RNA genome segments of awild-type artificial recombinant virus and an artificial recombinantvirus having a deletion mutant of the NSP1 gene.

FIG. 6 shows the structures of a wild-type NSP1 gene and an NSP1 genehaving a luciferase gene insertion.

FIG. 7 shows the results of plaque assay of an artificial recombinantrotavirus expressing luciferase (left) and the results of luminescencedetection in plaques (right).

FIG. 8 shows the results confirming that the replication capability ofMammalian orthoreovirus (MRV) and rotavirus (RV) was enhanced by usinghost cells transfected with a FAST protein expression vector.

FIG. 9 shows the structure of plasmid p3E5.

FIG. 10 shows the structure of plasmid pCAGGS.

FIG. 11 shows the results of SDS-PAGE of the RNA genome segments of anartificial recombinant simian rotavirus having the NSP4 segment of thehuman rotavirus RNA genome, the RNA genome segments of a wild-type humanrotavirus, and the RNA genome segments of a wild-type simian rotavirus.

FIG. 12 shows the results of the viral proliferation inhibitory effectof an anti-rotavirus drug, ribavirin, assessed based on luminescenceintensity measured in a test using a luciferase-expressing artificialrecombinant rotavirus.

FIG. 13 shows the comparison of the replication capabilities of anartificial recombinant rotavirus having a mutation in NSP4 and awild-type artificial recombinant rotavirus.

FIG. 14 shows the structures of various forms of NSP1 genes having aZsGreen gene insertion. The 1st row shows an NSP1 gene having no basedeletion, and the 2nd to 4th rows show NSP1 genes having partial basedeletions.

FIG. 15 shows the retention rate (ZsGreen expression level) of theZsGreen gene after serial passage of four different ZsGreen-expressingartificial recombinant rotaviruses each produced using a genome segmentexpression vector containing the corresponding NSP1 gene having aZsGreen gene insertion shown in FIG. 14 .

DESCRIPTION OF EMBODIMENTS

Method for Producing an Artificial Recombinant Virus

The present invention provides a method for producing an artificialrecombinant virus of the family Reoviridae (hereinafter, referred to asthe “production method of the present invention”). The production methodof the present invention comprises the steps of:

(1) introducing a FAST protein expression vector and/or a capping enzymeexpression vector into host cells;

(2) introducing a vector containing expression cassettes for individualRNA genome segments of a virus or introducing a set of single-strandedRNA transcripts from the expression cassettes into host cells; and

(3) culturing the host cells.

The viruses in the family Reoviridae possess a linear double-strandedRNA (dsRNA) genome consisting of 10 to 12 segments and have anicosahedral virion of 60 to 80 nm in diameter. The viruses in the familyReoviridae include members of the genus Orthoreovirus such as Mammalianorthoreovirus, Nelson Bay reovirus and Avian reovirus; members of thegenus Orbivirus such as African horse sickness virus and bluetonguevirus; members of the genus Rotavirus such as rotaviruses; members ofthe genus Coltivirus such as Colorado tick fever virus; members of thegenus Aquareovirus such as Aquareovirus A; members of the genusCypovirus such as cytoplasmic polyhedrosis virus; members of the genusFijivirus such as Southern rice black-streaked dwarf virus; members ofthe genus Phytoreovirus such as rice dwarf virus; and members of thegenus Oryzavirus such as rice ragged stunt virus. The production methodof the present invention is particularly preferably applied to anartificial recombinant Mammalian orthoreovirus or rotavirus.

The expression cassettes for individual RNA genome segments of a virusare not particularly limited as long as the expression cassettes aredesigned to allow the expression of single-stranded plus strand RNA(viral mRNA) to serve as a template of the segmented genome dsRNA of thevirus. Preferably, each expression cassette is composed of, from theupstream, an RNA polymerase promoter, a DNA encoding an RNA genomesegment (a cDNA of an RNA genome segment) and a DNA encoding aself-cleaving ribozyme. In the case where the RNA polymerase promoter isT7 promoter, the expression cassette contains a T7 terminator sequence.In the case where the RNA polymerase promoter is a polymerase Ipromoter, the expression cassette contains a terminator sequencecorresponding to the promoter. In the case where the RNA polymerasepromoter is a polymerase II promoter, the expression cassette contains apolyadenylation signal sequence.

Each of the expression cassettes for individual RNA genome segments of avirus may be composed only of an RNA polymerase promoter and a DNAencoding an RNA genome segment (a cDNA of an RNA genome segment). Avector containing such an expression cassette is cleaved at the 3′ endof the DNA encoding an RNA genome segment with, for example, arestriction enzyme, resulting in a linear vector. The linear vectorencodes the viral genome in the 3′ end region, and therefore can be usedwithout the risk of transcription of the original sequence of thevector.

The cDNA of each RNA genome segment can be obtained by RNA extractionfrom the virus, followed by RT-PCR using the extracted dsRNA as atemplate. The primer sets used for RT-PCR can be designed to be specificto the corresponding RNA genome segments based on their nucleotidesequences. Information on the nucleotide sequence of each RNA genomesegment is available from known databases (GenBank etc.). Alternatively,the nucleotide sequence of each RNA genome segment may be determined bya known method using a commercial sequencer.

Preferable examples of the RNA polymerase promoter include T7 promoter,polymerase I promoter, and polymerase II promoters including CAGpromoter and CMV promoter. Preferred is T7 promoter.

The self-cleaving ribozyme can be selected as appropriate from knownself-cleaving ribozymes. Examples of the self-cleaving ribozyme includea hammerhead ribozyme, a hairpin ribozyme, ribonuclease P subunit M1, ahepatitis D virus (HDV) ribozyme and a Varkud satellite ribozyme.Preferred is an HDV ribozyme.

Preferable examples of the vector into which the RNA genome segmentexpression cassette is to be inserted include known cloning vectors,known mammalian cell expression vectors and various types of known viralvectors (a vaccinia virus vector, an adenovirus vector, anadeno-associated virus vector, a retrovirus vector, a lentivirus vector,etc.).

In the case where each vector contains a single RNA genome segmentexpression cassette, the vectors as many as the viral genome segmentsrepresent one set. A polycistronic vector containing two or more RNAgenome segment expression cassettes may be used. The number of the RNAgenome segment expression cassettes contained in a single vector is notlimited, and one vector may contain all the RNA genome segmentexpression cassettes. In view of the efficiency of artificialrecombinant virus production, the number of vectors is preferablysmaller.

The single-stranded RNA (ssRNA) to be introduced into host cells is aplus strand RNA and can be obtained by in vitro transcription from thecorresponding RNA genome segment expression cassette. The in vitrotranscription can be performed, for example, using a commercial reagent(e.g., in vitro Transcription T7 Kit (Takara Bio) etc.). After in vitrotranscription, the obtained RNA is desirably capped using a cap analog(e.g., Ribo m7G Cap Analog (Promega) etc.) before use. The set of ssRNAsinclude ssRNAs as many as the viral genome segments.

Examples of the FAST (fusion-associated small transmembrane) proteinthat can be used include FAST proteins of known fusogenic reovirusesbelonging to the genus Orthoreovirus of the family Reoviridae and FASTproteins of yet-to-be-isolated viruses. Specific examples include NelsonBay reovirus p10 (GenBank ACCESSION: BAJ52806), Avian reovirus p10(GenBank ACCESSION: AGO32037), Broome reovirus p13 (GenBank ACCESSION:YP_003717780), Reptilian reovirus p14 (GenBank ACCESSION: AAP03134),Baboon reovirus p15 (GenBank ACCESSION: YP_004769555), grass carpreovirus p16 (GenBank ACCESSION: ABV01045), and Atlantic salmon reovirusp22 (GenBank ACCESSION: ACN38055). Preferred are Nelson Bay reovirus p10and Avian reovirus p10.

The FAST protein expression vector can be prepared by inserting a geneencoding any of the above FAST proteins into a known mammalian cellexpression vector, exemplified by plasmid pCAGGS (see FIG. 10 ), or aknown viral vector. The nucleotide sequence data of the FASTprotein-encoding gene can be obtained from the nucleotide sequence dataof the viral genome of interest. The nucleotide sequence data of theviral genome may be the ones registered in known databases (GenBanketc.). The nucleotide sequence of the gene encoding Nelson Bay reovirusp10 may be, for example, the nucleotide sequence of SEQ ID NO: 26. Thenucleotide sequence of the gene encoding Avian reovirus p10 may be, forexample, the nucleotide sequence of SEQ ID NO: 27.

Instead of the FAST protein expression vector, a single-stranded plusstrand RNA encoding the FAST protein may be used. The single-strandedplus strand RNA encoding the FAST protein can be obtained, for example,by in vitro transcription from the FAST protein expression vector. Thein vitro transcription can be performed, for example, using a commercialreagent (e.g., in vitro Transcription T7 Kit (Takara Bio) etc.). Afterin vitro transcription, the obtained RNA is desirably capped using a capanalog (e.g., Ribo m7G Cap Analog (Promega) etc.) before use.

The capping enzyme is not particularly limited as long as the enzyme cancatalyze mRNA capping in the cytoplasm. For example, capping enzymes ofDNA or RNA viruses which replicate in the cytoplasm of host cells canpreferably be used. The capping enzymes of DNA viruses which replicatein the cytoplasm of host cells include, for example, capping enzymesencoded by viruses in the family Poxviridae and capping enzymes encodedby viruses in the family Asfarviridae. The capping enzymes of RNAviruses which replicate in the cytoplasm of host cells include, forexample, nsp1 protein of viruses in the family Togaviridae. Preferredare capping enzymes encoded by viruses in the family Poxviridae orAsfarviridae. Among the capping enzymes encoded by viruses in the familyPoxviridae, vaccinia virus capping enzymes can preferably be used. Amongthe capping enzymes encoded by viruses in the family Asfarviridae,African swine fever virus capping enzyme NP868R can preferably be used.In the case where a vaccinia virus capping enzyme is used, expressionvectors for the capping enzyme can be prepared by inserting a gene (DiR)encoding the large subunit of the capping enzyme (GenBank ACCESSION:YP_232988) and a gene (D12L) encoding the small subunit of the cappingenzyme (GenBank ACCESSION: YP_232999) into separate vectors such asknown mammalian cell expression vectors exemplified by plasmid pCAGGS(see FIG. 10 ) and known viral vectors. The nucleotide sequence of thegene encoding the large subunit of a vaccinia virus capping enzyme maybe, for example, the nucleotide sequence of SEQ ID NO: 29. Thenucleotide sequence of the gene encoding the small subunit of a vacciniavirus capping enzyme may be, for example, the nucleotide sequence of SEQID NO: 30. A polycistronic vector containing capping enzyme subunitexpression cassettes together with a FAST protein expression cassettemay be used.

Instead of the capping enzyme expression vectors, single-stranded plusstrand RNAs encoding the subunits of the capping enzyme may be used. Thesingle-stranded plus strand RNAs encoding the subunits of the cappingenzyme can be obtained, for example, by in vitro transcription from theexpression vectors for the subunits of the capping enzyme. The in vitrotranscription can be performed, for example, using a commercial reagent(e.g., in vitro Transcription T7 Kit (Takara Bio) etc.). After in vitrotranscription, the obtained RNAs are desirably capped using a cap analog(e.g., Ribo m7G Cap Analog (Promega) etc.) before use.

The host cells are preferably cells with high susceptibility to virusesin the family Reoviridae and high transfection efficiency. Examples ofsuch cells include but not limited to BHK cells, MA104 cells, COS7cells, CV1 cells, Vero cells, L929 cells, 293T cells and A549 cells. Inaddition, modified cells derived from any of the above cells (newlycloned cells, foreign gene-transfected cells, etc.) can also preferablybe used as the host cells.

In the case where T7 promoter is used as the promoter of each RNA genomesegment expression cassette, recombinant T7 RNA polymerase-expressingcells can be used as the host cells. The recombinant T7 RNApolymerase-expressing cells can be prepared, for example, bytransfecting appropriate host cells with a mammalian cell expressionvector containing a gene encoding T7 RNA polymerase (GenBank ACCESSION:ADJ00046) and selecting cells stably expressing T7 RNA polymerase by adrug-based selection technique or the like. Alternatively, cellstransiently or permanently expressing a recombinant T7 RNA polymerasecan be prepared, for example, by infecting appropriate host cells with aviral vector (a vaccinia virus vector, an adenovirus vector, anadeno-associated virus vector, a retrovirus vector, a lentivirus vectoror the like) containing a gene encoding T7 RNA polymerase. Thenucleotide sequence of the gene encoding T7 RNA polymerase may be, forexample, the nucleotide sequence of positions 894 to 3545 of “T7 RNApolymerase vector pGemT7cat” (GenBank ACCESSION: HM049174). Anotherexample of the recombinant T7 RNA polymerase-expressing cells may beknown recombinant T7 RNA polymerase-expressing cells (e.g., BHK/T7-9:Ito, N et al., (2003), Microbiology and immunology 47, 613-617).

The introduction of a FAST protein expression vector and/or a cappingenzyme expression vector into host cells in step (1) and theintroduction of a vector containing expression cassettes for individualRNA genome segments of a virus or of a set of single-stranded RNAtranscripts from the expression cassettes into host cells in step (2)can be performed using a known transfection method, such aselectroporation, the calcium phosphate method, the liposome method orthe DEAE dextran method. Commercial transfection reagents (e.g.,TransIT-LT1 (trade name, Mirus) etc.) can also be used for theintroduction.

In the production method of the present invention, step (1) and step (2)may be performed separately or concurrently. In the case where step (1)and step (2) are performed separately, step (1) may precede or followstep (2). Step (1) and step (2) are preferably performed concurrently.That is, the method of the present invention preferably comprises thesteps of:

(I) introducing a FAST protein expression vector and/or a capping enzymeexpression vector into host cells, concurrently with introducing avector containing expression cassettes for individual RNA genomesegments of a virus or introducing a set of single-stranded RNAtranscripts from the expression cassettes into the host cells; and

(II) culturing the host cells.

The host cells into which the FAST protein expression vector and/or thecapping enzyme expression vector have been introduced may transiently orpermanently express the FAST protein and/or the capping enzyme. In thecase where the recombinant T7 RNA polymerase-expressing cells asdescribed above are used as the host cells, the transfected host cellsmay be cells permanently expressing a FAST protein and/or a cappingenzyme in addition to T7 RNA polymerase. The cells permanentlyexpressing a FAST protein and/or a capping enzyme can be prepared byintroducing a FAST protein expression vector and/or a capping enzymeexpression vector into cells and selecting cells stably expressing aFAST protein and/or a capping enzyme by a drug-based selection techniqueor the like. The permanently expressing cells may be cells whichconstitutively express a FAST protein and/or a capping enzyme, or cellswhich express a FAST protein and/or a capping enzyme in a controlledmanner, for example, under a Tet on/off system etc. Alternatively, cellstransiently or permanently expressing a FAST protein and/or a cappingenzyme can be prepared, for example, by infecting appropriate host cellswith a viral vector (a vaccinia virus vector, an adenovirus vector, anadeno-associated virus vector, a retrovirus vector, a lentivirus vectoror the like) containing a gene encoding a FAST protein and/or a geneencoding a capping enzyme.

The amount of the nucleic acid used for transfection is preferablyselected as appropriate for the size of the culture plate used, the typeof the host cells, the seeding cell number, etc. For example, in thecase where BHK cells stably expressing T7 RNA polymerase are seeded ashost cells at 8×10⁵ cells/well on a 6-well plate on the previous day oftransfection, the DNA amount of each RNA genome segment expressionvector is preferably 0.5 to 1.0 μg, the DNA amount of the FAST proteinexpression vector is preferably 0.002 to 0.02 μg, and the DNA amount ofthe capping enzyme expression vector is preferably 0.5 to 1.0 μg. Forexample, in the case where BHK cells stably expressing T7 RNA polymeraseare seeded as host cells at 4×10⁵ cells/well on a 12-well plate on theprevious day of transfection, the DNA amount of each RNA genome segmentexpression vector is preferably 0.25 to 0.5 μg, the DNA amount of theFAST protein expression vector is preferably 0.0001 to 0.01 μg, and theDNA amount of the capping enzyme expression vector is preferably 0.25 to0.5 μg.

For the culture of the host cells, a medium suitable for the host cellsis selected and used. Cytopathic changes of the host cells indicate thatthe artificial recombinant virus has been produced. The medium and cellson a plate or in a well in which cytopathic changes have been observedare harvested to prepare a cell lysate, which may be used as a virussample. Alternatively, a virus sample can be prepared by isolation ofthe virus from the cell lysate by plaque assay, followed by mass cultureand viral particle purification. The viral particle purification can beperformed by known methods (e.g., cesium chloride density gradientcentrifugation etc.).

After culturing the host cells for several days, regardless of thepresence or absence of cytopathic changes, a cell lysate may be preparedas described above and added to other cells for virus passage. The cellsto which the cell lysate is added are preferably highlyvirus-susceptible cells, and more preferably cells with highsusceptibility to the virus of interest. For example, for production ofan artificial recombinant rotavirus, MA104 cells or CV1 cells arepreferable. Cytopathic changes of the cells cultured with the celllysate indicate that the artificial recombinant virus has been produced.

Several days after the transfection of the set of vectors etc. into hostcells, highly virus-susceptible cells as described above may beadditionally seeded on the culture plate or in the wells containing thehost cells and co-cultured with the host cells. In the case of suchco-culture, the seeding cell number of the additional cells ispreferably about ⅕ to 1/20 of the cells having been subjected to thetransfection. After cell addition, culture is continued in atrypsin-containing (e.g., about 0.5 μg/mL) serum-free medium for about 3to 5 days. Then, a cell lysate is prepared and added to highlyvirus-susceptible cells for passage. The highly virus-susceptible cellsare cultured in the same trypsin-containing serum-free culture medium asabove. Cytopathic changes of the cells indicate that the artificialrecombinant virus has been produced. The medium and cells on a plate orin a well in which cytopathic changes have been observed are harvestedto prepare a cell lysate, which may be used as a virus sample.Alternatively, a virus sample can be prepared by isolation of the virusfrom the cell lysate by plaque assay, followed by mass culture and viralparticle purification. The viral particle purification can be performedby known methods (e.g., cesium chloride density gradient centrifugationetc.).

According to the production method of the present invention, anartificial recombinant virus having a mutation introduced in at leastone RNA genome segment, an artificial recombinant virus having a foreigngene inserted in at least one RNA genome segment, or an artificialrecombinant virus having a mutation introduced in at least one RNAgenome segment and a foreign gene inserted in at least one RNA genomesegment can be produced. Such artificial recombinant viruses can beproduced by introducing a desired mutation into an expression cassettefor the RNA genome segment and/or by inserting a desired foreign geneinto an expression cassette for the RNA genome segment. The mutationintroduction and foreign gene insertion into an expression cassette forthe RNA genome segment can be performed by known gene recombinationtechniques.

The present inventors have successfully produced an artificialrecombinant rotavirus which has a deletion mutation in rotavirus NSP1and is capable of autonomous proliferation, an artificial recombinantrotavirus which has a deletion mutation in rotavirus NSP3 and is capableof autonomous proliferation, and an artificial recombinant rotaviruswhich has a mutation resulting in amino acid substitution in rotavirusNSP4 and is capable of autonomous proliferation. According to theproduction method of the present invention, an artificial recombinantrotavirus incapable of autonomous proliferation can be produced bypartial deletion of a viral protein gene essential for proliferation.More specifically, an artificial recombinant virus incapable ofautonomous proliferation can be produced using host cells modified toexpress a mutant form of a viral protein essential for proliferation dueto partial deletion of the corresponding gene. Such an artificialrecombinant virus can proliferate only in cells expressing a normal formof the viral protein. Therefore, this type of artificial recombinantvirus can be applied to the production of single-round infectiousvirus-like particles and is expected to be useful as a vaccine.Moreover, an artificial recombinant virus as an attenuated vaccinecandidate can also be produced by introducing a mutation into a knownviral protein gene associated with the degree of virulence.

In addition, the present inventors have successfully produced anartificial recombinant rotavirus expressing luciferase by inserting aluciferase gene (Nluc gene) into the rotavirus NSP1 gene. Moreover, thepresent inventors have successfully produced an artificial recombinantrotavirus expressing ZsGreen by inserting a green fluorescent proteingene (ZsGreen gene) into the rotavirus NSP1 gene. The foreign gene canbe inserted into any genome segment of a rotavirus. The foreign gene isnot limited to a gene of 500 bp or longer, such as a Nluc gene (SEQ IDNO: 31) or a ZsGreen gene (SEQ ID NO: 33). For example, a short peptidecan be expressed in a fusion protein with a viral protein. In the casewhere the artificial recombinant rotavirus has two or more foreigngenes, the two or more foreign genes may be inserted in separate genomesegments or inserted in one genome segment. The combination of themutation and the foreign gene in the genome segments is also notparticularly limited and can be selected as appropriate.

An expression vector for the foreign gene preferably contains a genomesegment expression cassette having a partial deletion in the rotavirusNSP1 gene and an insertion of the foreign gene in the rotavirus NSP1gene. The insertion site of the foreign gene is not particularly limitedand is preferably within a region which starts at about 30 to 200 basesdownstream from the 5′ end (including the untranslated region) of theNSP1 gene and ends at about 30 to 200 bases upstream from the 3′ end(including the untranslated region) of the NSP1 gene. More preferably,the insertion site is in the region of about 80 to 150 bases from the 5′end (including the untranslated region) of the NSP1 gene. Still morepreferably, the insertion site is in the region of about 100 to 130bases from the 5′ end (including the untranslated region) of the NSP1gene. The deletion region in the NSP1 gene is particularly not limited,but is preferably downstream the insertion site of the foreign gene. The3′-end region (including the untranslated region) of the NSP1 gene,however, is preferably retained. Preferably, a region of at least about30 bases or more, about 50 bases or more, about 100 bases or more, orabout 200 bases or more from the 3′ end of the NSP1 gene is retained.The number of bases deleted is not particularly limited and may be 1550bases or less, 1200 bases or less, 1000 bases or less, 800 bases orless, 700 bases or less, 600 bases or less, 500 bases or less, or 400bases or less. In addition, the number of bases deleted may be 100 basesor more, 200 bases or more, or 300 bases or more. With such a foreigngene expression vector, an artificial recombinant rotavirus which stablyretains a foreign gene over a long period of time and stably expressesthe foreign gene product can be produced.

The artificial recombinant virus having a mutation and the virusexpressing a foreign gene, each of which is produced by the productionmethod of the present invention, are useful for functional analysis ofviral proteins and for the development of vaccines and vaccine vectors.The artificial recombinant virus having a mutation and the virusexpressing a foreign gene can be used also as vaccines.

The production method of the present invention can enhance theefficiency of artificial recombinant rotavirus production byoverexpressing a rotavirus NSP2 gene product and/or a rotavirus NSP5gene product in the host cells. Either or both of the NSP2 gene productand the NSP5 gene product may be overexpressed in the host cells.Preferably, both the NSP2 gene product and the NSP5 gene product areoverexpressed in the host cells.

The overexpression of the NSP2 gene product and/or the NSP5 gene productin the host cells can be effected by preparing a vector expressing theNSP2 gene product (hereinafter referred to as an “NSP2 expressionvector”) and a vector expressing the NSP5 gene product (hereinafterreferred to as an “NSP5 expression vector”) and introducing either orboth of them into the host cells. The NSP2 expression vector and theNSP5 expression vector can be prepared, for example, by inserting theNSP2 gene (GenBank ACCESSION: LC178571, SEQ ID NO: 18) and the NSP5 gene(GenBank ACCESSION: LC178574, SEQ ID NO: 21) into separate vectors suchas known mammalian cell expression vectors exemplified by plasmid pCAGGS(see FIG. 10 ) and known viral vectors.

The NSP2 gene to be inserted into the NSP2 expression vector and theNSP5 gene to be inserted into the NSP5 expression vector may be from thestrain of an artificial recombinant rotavirus to be produced, or from arotavirus of a different genotype, a different serotype or a differentanimal (a human, a monkey, a horse, a bird, a dog, a pig, a cow, amouse, a rat, a rabbit, etc.). A polycistronic vector containing theNSP2 expression cassette together with the NSP5 expression cassette maybe used.

Instead of the NSP2 expression vector, a single-stranded plus strand RNAencoding NSP2 may be used. Similarly, instead of the NSP5 expressionvector, a single-stranded plus strand RNA encoding NSP5 may be used.These single-stranded plus strand RNAs can be obtained, for example, byin vitro transcription from the NSP2 expression vector and the NSP5expression vector. The in vitro transcription can be performed, forexample, using a commercial reagent (e.g., in vitro Transcription T7 Kit(Takara Bio) etc.). After in vitro transcription, the obtained RNAs aredesirably capped using a cap analog (e.g., Ribo m7G Cap Analog (Promega)etc.) before use.

The overexpression of the NSP2 gene product and/or the NSP5 gene productin the host cells can be effected without using the NSP2 expressionvector and/or the NSP5 expression vector, more specifically, byincreasing the amount(s) of an RNA genome segment expression vector forexpressing segment 8 (NSP2 gene) (segment 8 expression vector) and/or anRNA genome segment expression vector for expressing segment 11 (NSP5gene) (segment 11 expression vector) introduced into the host cells ascompared with those of RNA genome segment expression vectors forexpressing segments other than segment 8 or 11. The DNA amount(s) of thesegment 8 expression vector and/or the segment 11 expression vectorintroduced into the host cells are/is not particularly limited as longas each DNA amount is larger than those of the other RNA genome segmentexpression vectors. Each DNA amount may be about 1.5- to 10-fold larger,or about 2- to 5-fold larger than those of the other RNA genome segmentexpression vectors. See Table 2 in Example 2 for each rotavirus genomesegment.

Further, the present inventors have confirmed that an artificialrecombinant rotavirus can be produced even without using the host cellsexpressing a FAST protein and/or a capping enzyme (see Example 10). Thatis, the present invention provides a method for producing an artificialrecombinant rotavirus, the method comprising introducing a vectorcontaining expression cassettes for 11 individual rotavirus RNA genomesegments or introducing a set of 11 ssRNA transcripts from theexpression cassettes into host cells expressing neither a FAST proteinnor a capping enzyme, and culturing the host cells.

A first embodiment of this production method involves using host cellsinto which an NSP2 expression vector and/or an NSP5 expression vectorhave been introduced. A second embodiment thereof involves introducingonly a vector containing expression cassettes for 11 individualrotavirus RNA genome segments or introducing only a set of 11 ssRNAtranscripts from the expression cassettes into host cells. In the secondembodiment, it is preferable that the amount(s) of a segment 8expression vector and/or a segment 11 expression vector introduced intothe host cells are/is increased as compared with those of the rest ofthe expression vectors for 11 rotavirus RNA genome segments. The DNAamount(s) of the segment 8 expression vector and/or the segment 11expression vector introduced into the host cells are/is not particularlylimited as long as each DNA amount is larger than those of the other RNAgenome segment expression vectors. Each DNA amount may be about 1.5- to10-fold larger, or about 2- to 5-fold larger than those of the other RNAgenome segment expression vectors.

The rotavirus NSP2 and NSP5 are known to form a viral inclusion body ininfected cells and function to provide a site of viral replication (HuL, Crawford S, Hyser J, Estes M, and Prasad V (2012): Rotavirusnon-structural proteins: Structure and Function Current Opinion inVirology 2(4): 380-388.). A viral inclusion body is a structure found incommon among viruses in the family Reoviridae. For example, μNS encodedby the M3 gene of the genus Orthoreovirus of the Family Reoviridae(Mammalian orthoreovirus, Nelson Bay reovirus, Avian reovirus, etc.),σNS encoded by the S3 or S4 gene of the same genus as above, and NS2encoded by segment 8 of the genus Orbivirus of the Family Reoviridae(African horse sickness virus, bluetongue virus, etc.) are also known toform a viral inclusion body and function to provide a site of viralreplication (Thomas C P, Booth T F, Roy P (1990): Synthesis ofbluetongue virus-encoded phosphoprotein and formation of inclusionbodies by recombinant baculovirus in insect cells: it binds thesingle-stranded RNA species. Journal of General Virology, 71 (Pt 9):2073-2083.). Based on this knowledge, the present inventors used hostcells into which a μNS expression vector and a σNS expression vectorhave been introduced in the course of the production of an artificialrecombinant Mammalian orthoreovirus, and confirmed that this approachgreatly improved the efficiency of artificial recombinant virusproduction (see Example 13).

Therefore, the present invention provides a method for producing anartificial recombinant virus of the family Reoviridae, the methodcomprising

introducing a vector containing expression cassettes for individual RNAgenome segments of a virus or introducing a set of single-stranded RNAtranscripts from the expression cassettes into host cells;

overexpressing, in the host cells, a gene product involved in theformation of viral inclusion bodies in infected cells; and

culturing the host cells.

The host cells may be host cells expressing a FAST protein and/or acapping enzyme or host cells expressing neither a FAST protein nor acapping enzyme. Examples of the gene product involved in the formationof viral inclusion bodies in infected cells include μNS encoded by theM3 gene of a virus in the genus Orthoreovirus, σNS encoded by the S3 orS4 gene of a virus in the genus Orthoreovirus, and NS2 encoded bysegment 8 of the genus Orbivirus. One of these gene products may beused, and also two or more of them may be used in combination.

For the overexpression of a gene product involved in the formation ofviral inclusion bodies in infected cells, an expression vector for thedesired gene may be introduced into the host cells. Alternatively, avector containing an expression cassette for an RNA genome segmentencoding the desired gene may be introduced, into the host cells, in anincreased DNA amount as compared with those of the other RNA genomesegment expression vectors. The DNA amount of the vector containing anexpression cassette for an RNA genome segment encoding the desired genemay be about 1.5- to 10-fold larger, or about 2- to 5-fold larger thanthose of the other RNA genome segment expression vectors.

Artificial Recombinant Rotavirus and Vaccine

The present invention provides an artificial recombinant rotavirushaving a mutation resulting in functional suppression of at least oneselected from NSP1, NSP3 and NSP4. The present inventors havesuccessfully produced an artificial recombinant rotavirus having aC-terminal 108-amino-acid deletion in rotavirus NSP1. The C-terminalregion of NSP1 is known to play an important role in the suppression ofnatural immunity (Barro, M., and Patton, J. T. (2005). Rotavirusnonstructural protein 1 subverts innate immune response by inducingdegradation of IFN regulatory factor 3. Proceedings of the NationalAcademy of Sciences of the United States of America 102, 4114-4119.).Therefore, this artificial recombinant rotavirus is areplication-competent attenuated virus and is expected to be useful as avaccine. The present inventors have also produced an artificialrecombinant rotavirus having a deletion mutation in NSP3 and anartificial recombinant rotavirus having a mutation resulting in aminoacid substitution in NSP3, and have confirmed that artificialrecombinant rotaviruses having a mutation in NSP1, NSP3 or NSP4 are lessproliferative as compared with the wild-type artificial recombinantrotavirus. Therefore, the artificial recombinant rotaviruses having amutation in NSP3 or NSP4 are also replication-competent attenuatedviruses and are expected to be useful as vaccines. The present inventionalso includes a vaccine comprising an artificial recombinant rotavirushaving a mutation resulting in functional suppression of at least oneselected from NSP1, NSP3 and NSP4.

The present invention provides an artificial recombinant rotavirusexpressing a foreign gene. The foreign gene is not particularly limited,and for example, when the foreign gene encodes a vaccine antigen, theproduced artificial recombinant rotavirus can be used as a vaccine.Examples of the vaccine antigen include a Norovirus antigen, whichcauses oral or mucosal infections, an adenovirus antigen, a hepatitis Aantigen, a Sapovirus antigen, a hand-foot-and-mouth disease virusantigen, an enterovirus antigen, an HIV antigen, a Salmonella antigen, aCampylobacter antigen, a Vibrio parahaemolyticus antigen, an E. coliO-157 antigen, a cholera antigen, a typhoid antigen and a dysenteryantigen. These vaccine antigens may be epitope peptides thereof. Acombination of two or more of artificial recombinant rotavirusesexpressing different foreign vaccine antigens can compose a vaccine.

An artificial recombinant rotavirus expressing, as a foreign gene, oneor more genes encoding an antigen protein(s) of a different type orstrain of rotavirus (e.g., VP4, VP7, etc.) can be provided as apolyvalent rotavirus vaccine. The antigen protein may be an epitopepeptide thereof.

An artificial recombinant rotavirus expressing a reporter gene readilyallows the visualization of the amount of virus, and therefore, can beapplied to screening for new anti-rotavirus drugs. The reporter gene canbe selected as appropriate from known reporter genes. Preferableexamples include a luciferase gene, a GFP gene and an RFP gene.

The present invention provides an artificial recombinant reassortantrotavirus. A reassortant rotavirus refers to a rotavirus which has anovel genotypic composition resulting from recombination between thegenome segments of different types or strains of rotaviruses. Areassortant is also called a genetically reassorted strain. With theproduction method of the present invention, an artificial recombinantreassortant rotavirus between any two types of rotaviruses can bedesigned and produced. Exchange of gene segments between different viralstrains occurs also in natural infection and is considered as animportant evolutionary strategy in viruses with a segmented genome suchas rotaviruses. The artificial recombinant rotavirus reassortant betweendifferent types or strains of rotaviruses is useful for functionalanalysis of their genome segments and is also very useful as a vaccinecandidate. For example, a reassortant containing VP4 gene segments ofdifferent serotypes of rotaviruses or VP7 gene segments of differentserotypes of rotaviruses can be used as a bivalent rotavirus vaccine. Amixture of two or more of such reassortants can compose a multivalentrotavirus vaccine.

Method for Promoting Viral Replication

The present invention provides a method for promoting viral replication.The method of the present invention for promoting viral replicationcomprises infecting host cells expressing a FAST protein with a virusand culturing the host cells. The virus that infects host cells is notparticularly limited and is preferably a virus of the family Reoviridae.In particular, preferred are Mammalian orthoreovirus and rotaviruses.The host cells expressing a FAST protein can be prepared by introducinga FAST protein expression vector into appropriate host cells. The FASTprotein expression vector can be a FAST protein expression vector asdescribed above in the production method of the present invention. Thehost cells and the introduction method of the vector into the host cellscan also be the same as those described above in the production methodof the present invention. The host cells expressing a FAST protein maytransiently or permanently express the FAST protein.

The amount of the nucleic acid used for transfection is preferablyselected as appropriate for the size of the culture plate used, the typeof the host cells, the seeding cell number, etc. For example, in thecase where Vero cells are seeded as host cells at 8×10⁵ cells/well on a6-well plate on the previous day of transfection, the DNA amount of theFAST protein expression vector is preferably 0.002 to 0.02 μg. The DNAamount used for transfection can be changed as appropriate such that itis proportional to the seeding cell number suitable for a plate to beused.

The infection of host cells with a virus can be performed by adding avirus sample to a culture medium of the host cells. The infectious doseis not particularly limited, and the MOI is preferably 0.1 to 0.0001.The culture period is not particularly limited and is preferably 16 to48 hours.

With the method of the present invention for promoting viralreplication, proliferation (replication) of a virus with a lowproliferation (replication) capacity can be promoted. In addition, thismethod is useful for preparation of a high-titer viral stock.

EXAMPLES

Hereinafter, the present invention will be described in detail byexamples, but the present invention is not limited thereto.

Example 1: Improvement of Reverse Genetics System for MammalianOrthoreovirus

Mammalian orthoreovirus (MRV) has been extensively studied as a modelvirus of the family Reoviridae. An entirely plasmid-based reversegenetics (RG) system for MRV is the first system developed in the familyReoviridae (Non Patent Literature 3). For the purpose of improving RGsystems for the family Reoviridae, it was examined whether the use of aFAST protein encoded by a group of fusogenic reoviruses and a cappingenzyme encoded by vaccinia virus could enhance the efficiency ofartificial recombinant virus production.

Materials and Methods

(1) Viruses

Mammalian orthoreovirus strain type 1 Lang (hereinafter referred to as“MRV T1L”) was used. MRV T1L can be purchased from ATCC (ATCC VR-230).The gene names and GenBank accession numbers of 10 individual RNA genomesegments of MRV T1L are shown in Table 1.

TABLE 1 Sequences of genome segments of MRV T1L Gene name GenBankACCESSION SEQ ID NO L1 M24734 1 L2 AF378003 2 L3 AF129820 3 M1 AF4616824 M2 AF490617 5 M3 AF174382 6 S1 EF494445 7 S2 L19774 8 S3 M14325 9 S4M13139 10(2) Preparation of Plasmids Containing Expression Cassettes forIndividual RNA Genome Segments (RNA Genome Segment Expression Vectors)of MRV T1L

Plasmids containing cDNAs of the 10 individual RNA genome segments ofMRV T1L (L1 to L3, M1 to M3, S1 to S4) were prepared as described inreference 1 (Kobayashi et al., Virology. 2010 Mar. 15; 398(2):194-200).The specific procedure was as follows. The individual RNA genomesegments were amplified by RT-PCR from extracted viral dsRNA as atemplate using the respective specific primers designed based on thenucleotide sequence of each segment. The RT-PCR products (cDNAs of theindividual RNA genome segments) were individually cloned into p3E5EGFP(Watanabe et al., (2004), Journal of virology, 78, 999-1005) to yieldplasmids each having an expression cassette in which the cDNA of thedesired single RNA genome segment was flanked by a T7 promoter sequence(SEQ ID NO: 22) at the 5′ end and a hepatitis D virus (HDV) ribozymesequence (SEQ ID NO: 23) at the 3′ end, followed by a T7 terminatorsequence (SEQ ID NO: 24). Each of the obtained plasmids had a structurein which the cDNA encoding the desired single RNA genome segment wasinserted between the T7 promoter sequence and the HDV ribozyme sequence(between positions 30 and 31 of SEQ ID NO: 25) of plasmid p3E5 (3076 bp,SEQ ID NO: 25, shown in FIG. 9 ).

Next, an M2 expression cassette was inserted into a plasmid with clonedL1 (pT7-L1T1L) to yield a cistronic plasmid (pT7-L1-M2T1L). Similarly,an M2 expression cassette was inserted into a plasmid with cloned L2(pT7-L2T1L) to yield a cistronic plasmid (pT7-L2-M3T1L), and an S3expression cassette was inserted into a plasmid with cloned L3(pT7-L3T1L) to yield a cistronic plasmid (pT7-L3-S3T1L). Further,expression cassettes for S3, S4 and M1 were inserted into a plasmid withcloned S2 (pT7-S2T1L) to yield a tetracistronic plasmid(pT7-S1-S2-S4-M1T1L).

(3) Preparation of FAST Protein Expression Vector

A FAST protein expression vector was prepared by inserting theprotein-coding region DNA (SEQ ID NO: 26) of the Nelson Bay reovirus p10gene (see GenBank ACCESSION: AB908284) or the protein-coding region DNA(SEQ ID NO: 27) of the Avian reovirus p10 gene (see GenBank ACCESSION:AF218358) into plasmid pCAGGS (5699 bp, SEQ ID NO: 28, shown in FIG. 10, Matsuo et al., 2006, Biochem Biophys Res Commun 340(1): 200-208).These coding region DNAs were synthesized by custom gene synthesisservices (Eurofins Genomics) based on the nucleotide sequences of SEQ IDNOs: 27 and 28. These synthetic DNAs were individually inserted into theBglII restriction site of plasmid pCAGGS (between positions 1753 and1754 of SEQ ID NO: 28) to yield pCAG-p10 (Nelson Bay reovirus p10expression vector) and pCAG-ARVp10 (Avian reovirus p10 vector).

(4) Preparation of Capping Enzyme Expression Vectors

Capping enzyme expression vectors were prepared by inserting theprotein-coding region DNA of the vaccinia virus D1R gene (GenBankACCESSION: NC006998, positions 93948 to 96482, SEQ ID NO: 29) and theprotein-coding region DNA of the vaccinia virus D12L gene (GenBankACCESSION: NC006998, positions 107332 to 108195, SEQ ID NO: 30) into thesame plasmid pCAGGS as above. These coding region DNAs were synthesizedby custom gene synthesis services (Eurofins Genomics) based on thenucleotide sequences of SEQ ID NOs: 29 and 30. These synthetic DNAs wereindividually inserted into the BglII restriction site of plasmid pCAGGS(between positions 1753 and 1754 of SEQ ID NO: 28) to yield pCAG-D1R(expression vector for the vaccinia virus mRNA capping enzyme largesubunit) and pCAG-D12L (expression vector for the vaccinia virus mRNAcapping enzyme small subunit).

(5) Host Cells

BHK-T7/P5 cells, which stably express T7 RNA polymerase, were used. TheBHK-T7/P5 cells were prepared by transfecting BHK cells (Baby HamsterKidney Cells) with a plasmid pCAGGS having a T7 RNA polymerase-encodingDNA inserted downstream of the CAG promoter and subsequently culturingthe BHK cells in a puromycin-containing medium for selection.

(6) Production of Artificial Recombinant Virus

BHK-T7/P5 cells were seeded on 24-well culture plates at 2×10⁵cells/well on the previous day of transfection. The BHK-T7/P5 cells weretransfected with 0.4 μg each of the RNA genome segment expressionvectors (pT7-L1-M2T1L, pT7-L2-M3T1L, pT7-L3-S3T1L andpT7-S1-S2-S4-M1T1L); 0.05 μg, 0.005 μg or 0.0005 μg of the FAST proteinexpression vector (pCAG-p10 or pCAG-ARVp10); and 0.2 μg each of thecapping enzyme expression vectors (pCAG-D1R and pCAG-D12L) using atransfection reagent (TransIT-LT1 (trade name), Mirus). The transfectionreagent was used in a volume of 2 μL per microgram of DNA. The BHK-T7/P5cells were cultured in DMEM medium supplemented with 5% FBS, 100units/mL penicillin and 100 μg/mL streptomycin in an atmosphere of 5%CO₂ at 37° C. The medium and the cells were harvested 48 hours after thetransfection. The harvested medium and cells were repeatedlyfreeze-thawed 3 times and used as a virus sample for plaque assay, fromwhich the viral titer was determined.

(7) Plaque Assay

The plaque assay was performed in the following procedure. (a) Seedmouse L929 cells on 6-well culture plates at 1.2×10⁶ cells/well/2 mL ofMEM medium and culture the cells overnight. (b) Add 110 μL of the abovevirus sample to 1 mL of physiological saline containing gelatin and stirthe mixture to prepare a 10-fold diluted solution. Repeat this step toprepare serially diluted virus samples.

(c) Remove the medium from each well and add 100 μL/well of the seriallydiluted virus samples (two wells per sample). Incubate the plates atroom temperature for 60 minutes with occasional agitation.

(d) Add 3 mL/well of prewarmed 2×199 medium/agar (a mixture of equalamounts of 2% agarose and 2×199 medium) and continue incubation at 37°C. for 2 days.

(e) Two days after step (d), overlay 2 mL/well of 2×199 medium/agar andcontinue incubation at 37° C. for 4 days.

(f) Two days after step (e), overlay 2 mL/well of 2×199 medium/agarcontaining neutral red and continue incubation at 37° C. overnight.

(g) Count plaques.

Results

The results are shown in FIG. 1 . As compared with the viral titer fromthe cells transfected with only the 4 expression vectors for the RNAgenome segments of MRV T1L (pT7-L1-M2T1L, pT7-L2-M3T1L, pT7-L3-S3T1L andpT7-S1-S2-S4-M1T1L), the viral titer from the cells co-transfected withthe FAST protein expression vector (pCAG-p10, 0.005 μg) was about 600times higher. The viral titer from the cells co-transfected with thecapping enzyme expression vectors (pCAG-D1R and pCAG-D12L) was about 100times higher. The viral titer from the cells co-transfected with acombination of the FAST protein expression vector and the capping enzymeexpression vectors was about 1,200 times higher. These results show thatco-transfection of the RNA genome segment expression vectors with theFAST protein expression vector and/or the capping enzyme expressionvectors into the host cells greatly improves the efficiency ofartificial recombinant virus production. However, in the case where theDNA amount of the FAST protein expression vector transfected into thehost cells was 0.05 μg, the viral titer was undetectable, and in thecase of 0.0005 μg, great increase in viral titer was not observed.Therefore, in the case of co-expression with the FAST protein in hostcells, the DNA amount of the FAST protein expression vector transfectedhas to be adjusted so as to ensure an appropriate expression level ofthe FAST protein.

Also in the case of using pCAG-ARVp10 as the FAST protein expressionvector, the results similar to those in FIG. 1 were obtained (data notshown). In addition, the present inventors performed an experiment onthe production of an artificial recombinant virus of Mammalianorthoreovirus strain type 3 Dearing (MRV T3D) by co-transfection ofexpression vectors for the RNA genome segments of MRV T3D (see NonPatent Literature 3) with a FAST protein expression vector and/orcapping enzyme expression vectors into host cells. The results confirmedthat such co-transfection greatly improved the efficiency of artificialrecombinant virus production as with the case of MRV T1L.

The present inventors also performed an experiment on artificialrecombinant Nelson Bay reovirus production, and as a result, confirmedthat co-transfection of expression vectors for the RNA genome segmentsof Nelson Bay reovirus with capping enzyme expression vectors into hostcells greatly improved the efficiency of artificial recombinant NelsonBay reovirus production. Nelson Bay reovirus expresses a FAST proteinfrom its own p10 gene.

Example 2: Development of Rotavirus Reverse Genetics System

Materials and Methods

(1) Virus

Simian rotavirus strain SA11 was used. The present inventors previouslydetermined and registered the nucleotide sequences of all 11 RNA genomesegments of this virus strain. The names and GenBank accession numbersof the 11 individual RNA genome segments of the simian rotavirus strainSA11 (hereinafter referred to as “SA11”) used in the experiment beloware shown in Table 2.

TABLE 2 Sequences of genome segments of simian rotavirus SA11 GenomeGenBank SEQ ID segment Coding protein ACCESSION NO Segment 1 VP1(RNA-dependent LC178564 11 RNA polymerase) Segment 2 VP2 (RNA-bindingLC178565 12 protein) Segment 3 VP3 (Guanylyltransferase) LC178566 13Segment 4 VP4 (Hemagglutinin, LC178567 14 spike protein) Segment 5 NSP1(Immune LC178570 15 suppressive factor) Segment 6 VP6 (Inner capsid)LC178568 16 Segment 7 NSP3 (Translation LC178572 17 enhancer) Segment 8NSP2 (NTPase) LC178571 18 Segment 9 VP7(Outer capsid) LC178569 19Segment 10 NSP4 (Enterotoxin) LC178573 20 Segment 11 NSP5 (RNA synthesisaid) LC178574 21(2) Preparation of Plasmids Containing Expression Cassettes forIndividual RNA Genome Segments (RNA Genome Segment Expression Vectors)of SA11

Plasmids containing cDNAs of the 11 individual RNA genome segments ofSA11 were prepared. The specific procedure was as follows. Theindividual RNA genome segments were amplified by RT-PCR from extractedviral dsRNA as a template using the respective specific primers designedbased on the nucleotide sequence of each segment. The RT-PCR products(cDNAs of the individual RNA genome segments) were individually insertedbetween the T7 promoter sequence and the HDV ribozyme sequence (betweenpositions 30 and 31 of SEQ ID NO: 25) of plasmid p3E5 (3076 bp, SEQ IDNO: 25, shown in FIG. 9 ) to yield plasmids each containing anexpression cassette for the desired RNA genome segment. Each of theexpression cassettes for individual RNA genome segments had a structurein which the cDNA of the corresponding segment was flanked by a T7promoter sequence (SEQ ID NO: 22) at the 5′ end and a hepatitis D virus(HDV) ribozyme sequence (SEQ ID NO: 23) at the 3′ end, followed by a T7terminator sequence (SEQ ID NO: 24). The prepared plasmids (RNA genomesegment expression vectors) are designated as pT7-VP1SA11, pT7-VP2SA11,pT7-VP3SA11, pT7-VP4SA11, pT7-VP6SA11, pT7-VP7SA11, pT7-NSP1SA11,pT7-NSP2SA11, pT7-NSP3SA11, pT7-NSP4SA11 and pT7-NSP5SA11.

(3) Preparation of Plasmids Having Marker Mutation(s)

Marker mutation was introduced into pT7-NSP1SA11, pT7-NSP2SA11,pT7-NSP3SA11 and pT7-NSP4SA11 using KOD-Plus-Mutagenesis Kit (tradename, Toyobo). More specifically, T at position 1053 of the NSP1 gene(SEQ ID NO: 15) of pT7-NSP1SA11 was mutated to C, and T at position 1059of the same gene was mutated to C; A at position 409 of the NSP2 gene(SEQ ID NO: 18) of pT7-NSP2SA11 was mutated to T, and T at position 418of the same gene was mutated to C; A at position 406 of the NSP3 gene(SEQ ID NO: 17) of pT7-NSP3SA11 was mutated to G, and A at position 412of the same gene was mutated to T; and G at position 389 of the NSP4gene (SEQ ID NO: 20) of pT7-NSP4SA11 was mutated to A, and A of position395 of the same gene was mutated to G. These mutations yielded a plasmidhaving a BamHI recognition sequence at positions 1049 to 1054 of theNSP1 gene (SEQ ID NO: 15), a plasmid having an EcoRV recognitionsequence at positions 413 to 418 of the NSP2 gene (SEQ ID NO: 18), aplasmid having an EcoRI recognition sequence at positions 408 to 413 ofthe NSP3 gene (SEQ ID NO: 17), and a plasmid having a MluI recognitionsequence at positions 393 to 398 of the NSP4 gene (SEQ ID NO: 20)(designated as pT7-NSP1SA11/BamHI, pT7-NSP2SA11/EcoRV,pT7-NSP3SA11/EcoRI and pT7-NSP4SA11/MluI, respectively) (see FIG. 2 ).

(4) FAST Protein Expression Vector

The FAST protein expression vector used was pCAG-p10 (Nelson Bayreovirus p10 expression vector), which was prepared in Example 1.

(5) Capping Enzyme Expression Vector

The capping enzyme expression vectors used were pCAG-D1R (expressionvector for the vaccinia virus mRNA capping enzyme large subunit) andpCAG-D12L (expression vector for the vaccinia virus mRNA capping enzymesmall subunit), both of which were prepared in Example 1.

(6) Host Cells

The host cells used were the same as those in Example 1, namelyBHK-T7/P5 cells, which stably express T7 RNA polymerase.

(7) Production of Artificial Recombinant Viruses

For production of a wild-type artificial recombinant virus, the 11 RNAgenome segment expression vectors prepared in the above (2) were used.For production of an artificial recombinant virus (rsSA11) having onemarker mutation, pT7-NSP4SA11/MluI was used instead of pT7-NSP4SA11. Forproduction of an artificial recombinant virus (rsSA11-3) having 3 markermutations, pT7-NSP1SA11/BamHI was used instead of pT7-NSP1SA11,pT7-NSP2SA11/EcoRV was used instead of pT7-NSP2SA11, andpT7-NSP3SA11/EcoRI was used instead of pT7-NSP3SA11.

BHK-T7/P5 cells were seeded on 6-well culture plates at 8×10⁵ cells/wellon the previous day of transfection. The BHK-T7/P5 cells weretransfected with 0.8 μg each of the 11 RNA genome segment expressionvectors; 0.015 μg of the FAST protein expression vector (pCAG-p10); and0.8 μg each of the capping enzyme expression vectors (pCAG-D1R andpCAG-D12L) using a transfection reagent (TransIT-LT1 (trade name),Mirus). The transfection reagent was used in a volume of 2 μL permicrogram of DNA. The BHK-T7/P5 cells were cultured in DMEM mediumsupplemented with 5% FBS, 100 units/mL penicillin and 100 μg/mLstreptomycin in an atmosphere of 5% CO₂ at 37° C. The medium and thecells were harvested 48 hours after the transfection. The harvestedmedium and cells were repeatedly freeze-thawed 3 times to prepare a celllysate, and the cell lysate was added to monkey MA104 cells (ATCCCRL-2378.1) for passage. More specifically, about 0.5 mL of the celllysate was added to confluent MA104 cells on 12-well plates in thepresence of 0.5 μg/mL trypsin. The MA104 cells were cultured in DMEMmedium without FBS. In the case where the cells showed cytopathicchanges during the 7 days of culture after the passage, artificialrecombinant virus production was judged as successful. In this example,cytopathic changes were observed in the cells transfected with theexpression vectors for wild-type SA11, rsSA11 or rsSA11-3 production,and therefore, the production of each type of artificial recombinantrotavirus was judged as successful.

(8) Confirmation of Marker Mutation

The medium and cells in the wells in which cytopathic changes were shownwere harvested and then repeatedly freeze-thawed 3 times to prepare acell lysate. From the cell lysate containing wild-type SA11, rsSA11 orrsSA11-3, viral genome RNA was extracted using the Trizol reagent(Thermo Scientific). Using the extracted RNA as a template, RT-PCR wasperformed with specific primers designed based on the nucleotidesequences of the RNA genome segments. SuperScript III ReverseTranscriptase (Thermo Scientific) was used as the reverse transcriptase.The amplified products of NSP1, NSP2 and NSP3 of wild-type SA11 weredigested with BamHI, EcoRV and EcoRI, respectively. The amplifiedproducts of NSP1, NSP2 and NSP3 of rsSA11-3 were also digested in thesame manner. The digestion products were subjected to 1.2% agarose gelelectrophoresis. The amplified products of NSP4 of wild-type SA11 andrsSA11 were digested with MluI, and the digestion products weresubjected to 1.2% agarose gel electrophoresis.

Results

The results are shown in FIGS. 3A to 3D. FIG. 3A shows anelectrophoretic pattern of BamHI-digested amplified products ofwild-type SA11 NSP1 and rsSA11-3 NSP1. FIG. 3B shows an electrophoreticpattern of EcoRV-digested amplified products of wild-type SA11 NSP2 andrsSA11-3 NSP2. FIG. 3C shows an electrophoretic pattern ofEcoRI-digested amplified products of wild-type SA11 NSP3 and rsSA11-3NSP3. FIG. 3D shows an electrophoretic pattern of MluI-digestedamplified products of wild-type SA11 NSP4 and rsSA11-3 NSP4. The resultsconfirmed that the genome RNAs of rsSA11-3 and rsSA11 had markermutation(s) and was digested with the corresponding restrictionenzyme(s). Therefore, the viruses obtained using the rotavirus reversegenetics system of this example were proven to be artificial recombinantrotaviruses derived from the RNA genome segment expression vectors.

Example 3: Production of Artificial Recombinant Rotavirus Having aDeletion Mutation

An experiment was performed to examine the feasibility of the productionof an artificial recombinant rotavirus having a partial deletionmutation in NSP1, a suppressive factor against host innate immuneresponses.

Materials and Methods

(1) Preparation of Plasmid Having a Deletion Mutation in NSP1 Gene

A plasmid having a mutated NSP1 gene (see FIG. 4 ), which had a 299-basedeletion at positions 1192 to 1490 of the NSP1 gene (SEQ ID NO: 15), wasprepared from pT7-NSP1SA11 as a template using KOD-Plus-Mutagenesis Kit(trade name, Toyobo) and specific primers for the gene. This plasmid(designated as pT7-NSP1SA11ΔC108) expresses an NSP1 protein having adeletion of C-terminal 108 amino acids of the native NSP1.

(2) Production of Artificial Recombinant Viruses and Confirmation ofMutation

A deletion mutant of rotavirus (rsSA11/NSP1ΔC108) was produced in thesame manner as in Example 2 except that pT7-NSP1SA11ΔC108 was usedinstead of pT7-NSP1SA11 in the set of the 11 RNA genome segmentexpression vectors prepared in Example 2 (2). A wild-type artificialrecombinant virus was also produced in the same manner as in Example 2.The medium and MA104 cells in the wells in which cytopathic changes wereshown were harvested and then repeatedly freeze-thawed 3 times toprepare a cell lysate. From the cell lysate containing rsSA11/NSP1ΔC108or wild-type SA11, viral genome RNA was extracted and then subjected toSDS-PAGE.

Results

The results are shown in FIG. 5 . As is clear from FIG. 5 , the band ofeach RNA genome segment of rsSA11/NSP1ΔC108 was observed at the sameposition as the corresponding band of a wild-type artificial recombinantvirus, except for NSP1. The position of the band of NSP1ΔC108 wasdifferent from that of the wild-type counterpart, showing that theNSP1ΔC108 genome RNA is shorter than the wild-type counterpart. Sincethe C-terminal region of NSP1 is important for suppression of innateimmunity, the mutant rotavirus produced in this example is areplication-competent attenuated virus and can be a promising vaccinecandidate.

Example 4: Production of Luciferase-Expressing Rotavirus

An experiment was performed to examine the feasibility of the productionof a foreign gene-expressing rotavirus for use as a vaccine vector.

Materials and Methods

(1) Preparation of NSP1 Expression Plasmid Having a Luciferase GeneInsertion

The Nluc gene, which is a luciferase gene of Oplophorus gracilirostris,was used as the luciferase gene. The Nluc protein-coding region atpositions 815 to 1330 (SEQ ID NO: 31) of vector pNL1.1 (Promega, GenBankACCESSION: KM359774, 3817 bp) was amplified by PCR. The amplifiedproduct was inserted between positions 128 and 129 of the NSP1 gene (SEQID NO: 15) of pT7-NSP1SA11 to prepare an NSP1 gene expression plasmidhaving a luciferase gene insertion (designated as pT7-NSP1SA11-Nluc)(see FIG. 6 ).

(2) Production of Artificial Recombinant Virus and Confirmation ofLuciferase Expression

A luciferase-expressing rotavirus was produced in the same manner as inExample 2 except that pT7-NSP1SA11-Nluc was used instead of pT7-NSP1SA11in the set of the 11 RNA genome segment expression vectors prepared inExample 2 (2). After 7 days from passage in MA104 cells, the medium andthe cells were harvested and then freeze-thawed 3 times to prepare acell lysate. The cell lysate was subjected to plaque assay. For theplaque assay, CV-1 cells (ATCC CCL-70) were used. The CV-1 cells werecultured in DMEM medium without FBS. The MA104 cell lysate was seriallydiluted 10-fold, and each serial dilution was added to confluent CV-1cells on 12-well plates for viral infection. After incubation for 60minutes, the medium was removed, and DMEM medium containing 0.8% agarosegel and 0.5 μg/mL trypsin was overlaid on the cells. Four days afterviral infection, luminescence from plaques was examined. Morespecifically, the substrate stock solution of Nano-Glo Luciferase AssaySystem (trade name, Promega) was diluted about 500-fold with DMEM mediumwithout FBS and added to each well, and luminescence was detected withan in vivo imaging system (IVIS Spectrum, manufactured by Xenogen).Then, the cells were fixed with 10% formaldehyde and stained withcrystal violet to visualize plaques.

Results

The results are shown in FIG. 7 . The left panel is an image of plaquesvisualized by crystal violet staining of cells, and the right panel is aluminescent image of the same well. The positions of plaques were thesame to those of luminescent signals, showing that an artificialrecombinant rotavirus expressing a luciferase gene had been produced.These results demonstrate that the insertion of a foreign gene into therotavirus genome is feasible, and the artificial recombinant rotavirusobtained in this example can be used as a vaccine vector. In addition tothe NSP1 gene, the NSP3 gene (Montero H, Arias C F, Lopez S. RotavirusNonstructural Protein NSP3 Is Not Required for Viral Protein Synthesis.Journal of Virology. 2006; 80(18):9031-9038. doi:10.1128/JVI.00437-06.)can be used as the foreign gene insertion site in the production offoreign gene-expressing viruses capable of autonomous proliferation.

Example 5: Production of Artificial Recombinant Rotaviruses Using FASTProtein Expression Vector or Capping Enzyme Expression Vectors

Materials and Methods

The expression vectors for the 11 RNA genome segments of simianrotavirus strain SA11 produced in “Materials and methods” (2) of Example2, the FAST protein expression vector pCAG-p10 produced in “Materialsand methods” (3) of Example 1, the capping enzyme expression vectorspCAG-D1R and pCAG-D12L produced in “Materials and methods” (4) ofExample 1 were variously combined as shown in Table 3 and transfectedinto BHK-T7/P5 cells (see Example 1) to examine whether artificialrecombinant rotaviruses could be produced. The specific procedure was asfollows. BHK-T7/P5 cells were seeded on 12-well culture plates at 4×10⁵cells/well on the previous day of transfection. The BHK-T7/P5 cells weretransfected with the above vectors in the combinations and DNA amountsdescribed in Table 3 using a transfection reagent (TransIT-LT1 (tradename), Mirus). Two days later, MA104 cells (4×10⁴ cells/well) wereadded, and culture was continued for 3 days. The medium and the cellswere harvested and then repeatedly freeze-thawed 3 times to prepare acell lysate. About 0.5 mL of the cell lysate was added to confluentMA104 cells on 12-well plates in the presence of 0.5 μg/mL trypsin, andculture was continued for 7 days.

TABLE 3 Group A Group B Group C DNA amount DNA amount DNA amount Vectorname (μg) (μg) (μg) pT7-SA11-VP1 0.25 0.25 0.25 pT7-SA11-VP2 0.25 0.250.25 pT7-SA11-VP3 0.25 0.25 0.25 pT7-SA11-VP4 0.25 0.25 0.25pT7-SA11-VP5 0.25 0.25 0.25 pT7-SA11-VP6 0.25 0.25 0.25 pT7-SA11-VP70.25 0.25 0.25 pT7-SA11-NSP1 0.25 0.25 0.25 pT7-SA11-NSP2 0.25 0.25 0.25pT7-SA11-NSP3 0.25 0.25 0.25 pT7-SA11-NSP4 0.25 0.25 0.25 pT7-SA11-NSP50.25 0.25 0.25 pCAG-D1R 0.25 — 0.25 pCAG-D12L 0.25 — 0.25 pCAG-p10 — 0.001 0.001Results

Cytopathic changes were observed in all groups, namely group A, in whichonly the capping enzyme expression vectors were co-expressed with the 11RNA genome segment expression vectors; group B, in which only the FASTprotein expression vector was co-expressed with the 11 RNA genomesegment expression vectors; and group C, in which a combination of thecapping enzyme expression vectors and the FAST protein expression vectorwas co-expressed with the 11 RNA genome segment expression vectors. Thatis, co-expression of the 11 RNA genome segment expression vectors evenwith capping enzyme expression vectors only or a FAST protein expressionvector only allows the production of artificial recombinant rotaviruses.

Example 6: Enhancement of Replication Capacity of MammalianOrthoreovirus (MRV) and Rotavirus (RV) by FAST Protein

Materials and Methods

Confluent Vero cells on 24-well plates were transfected with the FASTprotein expression vector (pCAG-p10) or a pCAG empty vector in an amountof 0, 0.25, 0.5, 1 or 2 μg. TransIT-LT1 (trade name, Mirus) was used asthe transfection reagent. Two hours later, the medium was replaced withfresh DMEM with 5% FBS, and the Vero cells were infected with Mammalianorthoreovirus (MRV T1L) or simian rotavirus (SA11) at an MOI of 0.001.After viral adsorption at 37° C. for 1 hour, the cells were washed 6times with PBS. The MRV-infected cells were cultured in DMEM with 5%FBS, and the RV-infected cells were cultured in FBS-free DMEM. After 16hours of infection, the medium and the cells were harvested and thenfreeze-thawed 3 times to prepare a cell lysate. The cell lysate wassubjected to plaque assay. The plaque assay was performed in the samemanner as in Example 1.

Results

The results are shown in FIGS. 8A and 8B. FIG. 8A shows the results forMRV and FIG. 8B shows the results for RV. The replication capability ofMRV was enhanced as result of transfection of 0.5 μg or more of pCAG-p10as compared with the empty vector (mock). The replication capability ofRV was enhanced as result of transfection of 1 μg or more of pCAG-p10 ascompared with the empty vector (mock). These results show that viralreplication capability is improved by using FAST protein-expressingcells as host cells.

Example 7: Production of Mono-Reassortant Rotavirus Between SimianRotavirus and Human Rotavirus

An experiment was performed to examine the feasibility of the productionof an artificial recombinant rotavirus (SA11/KUNSP4) which was derivedfrom simian rotavirus and had a human rotavirus NSP4 gene as the NSP4gene segment.

Materials and Methods

(1) Human Rotavirus

Human rotavirus strain KU (Urasawa, S., Urasawa, T., Taniguchi, K., andChiba, S. (1984). Serotype determination of human rotavirus isolates andantibody prevalence in pediatric population in Hokkaido, Japan. Archivesof virology 81, 1-12.) was used.

(2) Preparation of Plasmid Having a Human Rotavirus NSP4 Gene

A plasmid containing a cDNA of the NSP4 segment of the KU RNA genome(GenBank ACCESSION: AB022772, SEQ ID NO: 32) was prepared. The specificprocedure was as follows. The NSP4 segment of the human rotavirus RNAgenome was amplified by RT-PCR from extracted viral dsRNA as a templateusing specific primers designed based on the nucleotide sequence of thesegment. The RT-PCR product (cDNA of the NSP4 segment of the RNA genome)was inserted between the T7 promoter sequence and the HDV ribozymesequence (between positions 30 and 31 of SEQ ID NO: 25) of plasmid p3E5(3076 bp, SEQ ID NO: 25, shown in FIG. 9 ) to yield a plasmid containingan expression cassette for the NSP4 segment of the RNA genome. Theexpression cassette for the NSP4 segment of the RNA genome had astructure in which the cDNA of the NSP4 segment was flanked by a T7promoter sequence (SEQ ID NO: 22) at the 5′ end and a hepatitis D virus(HDV) ribozyme sequence (SEQ ID NO: 23) at the 3′ end, followed by a T7terminator sequence (SEQ ID NO: 24). The prepared plasmid is designatedas pT7-NSP4KU.

(3) Production of Artificial Recombinant Virus and Confirmation ofMutation

An NSP4 mono-reassortant rotavirus (SA11/KUNSP4) was produced in thesame manner as in Example 2 except that pT7-NSP4KU was used instead ofpT7-NSP4SA11 in the set of the 11 RNA genome segment expression vectorsprepared in Example 2 (2). The medium and MA104 cells in the wells inwhich cytopathic changes were shown were harvested and then repeatedlyfreeze-thawed 3 times to prepare a cell lysate. Viral genome RNA wasextracted from SA11/KUNSP4 and then subjected to SDS-PAGE together withviral genome RNAs extracted from wild-type SA11 and wild-type KU.

Results

The results are shown in FIG. 11 . As is clear from FIG. 11 , the bandof each RNA genome segment of SA11/KUNSP4 was observed at the sameposition as the corresponding band of wild-type SA11, except for NSP4(“g10KU” in the figure). The band of the NSP4 segment of SA11/KUNSP4 wasobserved at the same position as the band of the NSP4 segment ofwild-type KU. These results show that the production method of thepresent invention allows the production of reassortant rotaviruses inwhich RNA genome segments of rotaviruses of various animal species arefreely combined.

Example 8: Screening Test for Anti-Rotavirus Drug UsingLuciferase-Expressing Rotavirus

An experiment was performed to examine the visualization of therotavirus proliferation inhibitory effect of a known anti-rotavirusdrug, ribavirin (Smee, D. F., Sidwell, R. W., Clark, S. M., Barnett, B.B., and Spendlove, R. S. (1982). Inhibition of rotaviruses by selectedantiviral substances: mechanisms of viral inhibition and in vivoactivity. Antimicrobial agents and chemotherapy 21, 66-73.) using theluciferase-expressing artificial recombinant rotavirus produced inExample 4.

Materials and Methods

CV-1 cells were seeded on 96-well culture plates at 1×10⁵ cells/well onthe previous day of infection. The CV-1 cells were infected withwild-type SA11 or the luciferase-expressing artificial recombinantrotavirus produced in Example 4 at an MOI of 0.001. After viraladsorption at 37° C. for 1 hour, the culture supernatant was removed,and DMEM (without FBS and with 0.5 μg/mL trypsin) containing 0, 1, 5,10, 50, 100 or 200 μM ribavirin (Sigma-Aldrich) was added. Incubationwas performed at 37° C. for 14 hours. After that, the substrate stocksolution of Nano-Glo Luciferase Assay System (trade name, Promega) wasadded to the culture medium, and luminescence was detected with an invivo imaging system (IVIS Spectrum, manufactured by Xenogen).

Results

The results are shown in FIG. 12 . As is clear from FIG. 12 , theluminescence intensity in the wells with ribavirin decreased in aribavirin concentration-dependent manner, and no luminescence wasobserved in the wells with ribavirin at concentrations of 50 μM or more.These results show that the extent of viral proliferation can be easilyvisualized using the luciferase-expressing artificial recombinantrotavirus. Therefore, the luciferase-expressing artificial recombinantrotavirus can be useful in screening for unidentified anti-rotavirusdrugs.

Example 9: Improvement of Rotavirus Reverse Genetics System (1)

To improve the rotavirus RG system by which artificial recombinantrotaviruses were successfully produced in Example 2, a system usingoverexpression of an NSP2 gene product and an NSP5 gene product wasevaluated in terms of the efficiency of artificial recombinant rotavirusproduction.

Materials and Methods

The RNA genome segment expression vectors, the FAST protein expressionvector and the capping enzyme expression vectors used in this examplewere the same as those in Example 2.

For preparation of an NSP2 expression vector and an NSP5 expressionvector, the protein-coding region DNA of the NSP2 gene of simianrotavirus SA11 (GenBank ACCESSION: LC178571, SEQ ID NO: 18) and theprotein-coding region DNA of the NSP5 gene of the same strain (GenBankACCESSION: LC178574, SEQ ID NO: 21) were individually inserted into theplasmid pCAGGS shown in FIG. 10 . These coding region DNAs weresynthesized by custom gene synthesis services (Eurofins Genomics). Thesesynthetic DNAs were individually inserted into the EcoRI restrictionsite of plasmid pCAGGS to yield pCAG-NSP2 and pCAG-NSP5.

The host cells used were BHK-T7/P5 cells, which stably express T7 RNApolymerase. The BHK-T7/P5 cells were prepared by transfecting BHK cells(Baby Hamster Kidney Cells) with a plasmid pCAGGS having a T7 RNApolymerase-encoding DNA inserted downstream of the CAG promoter andsubsequently culturing the BHK cells in an antibiotic-containing mediumfor selection.

BHK-T7/P5 cells were seeded on 24-well culture plates at 2×10⁵cells/well on the previous day of transfection. The BHK-T7/P5 cells weretransfected with the RNA genome segment expression vectors (pT7-VP1SA11,pT7-VP2SA11, pT7-VP3SA11, pT7-VP4SA11, pT7-VP6SA11, pT7-VP7SA11,pT7-NSP1SA11, pT7-NSP2SA11, pT7-NSP3SA11, pT7-NSP4SA11 andpT7-NSP5SA11); the FAST protein expression vector (pCAG-FAST p10); thecapping enzyme expression vectors (pCAG-D1R and pCAG-D12L); the NSP2expression vector (pCAG-NSP2); and the NSP5 expression vector(pCAG-NSP5) in the combinations and DNA amounts described in Table 4using a transfection reagent (TransIT-LT1 (trade name), Mirus). Thetransfection reagent was used in a volume of 2 μL per microgram of DNA.

TABLE 4 Group Group Group Group A DNA B DNA C DNA D DNA amount amountamount amount Vector name (μg) (μg) (μg) (μg) pT7-VP1SA11 0.125 0.1250.125 0.125 pT7-VP2SA11 0.125 0.125 0.125 0.125 pT7-VP3SA11 0.125 0.1250.125 0.125 pT7-VP4SA11 0.125 0.125 0.125 0.125 pT7-VP5SA11 0.125 0.1250.125 0.125 pT7-VP6SA11 0.125 0.125 0.125 0.125 pT7-VP7SA11 0.125 0.1250.125 0.125 pT7-NSP1SA11 0.125 0.125 0.125 0.125 pT7-NSP2SA11 0.1250.125 0.125 0.125 pT7-NSP3SA11 0.125 0.125 0.125 0.125 pT7-NSP4SA110.125 0.125 0.125 0.125 pT7-NSP5SA11 0.125 0.125 0.125 0.125 pCAG-FAST0.001 0.001 0.001 0.001 pCAG-D1R 0.125 0.125 0.125 0.125 pCAG-D12L 0.1250.125 0.125 0.125 pCAG-NSP2 — 0.125 — 0.125 pCAG-NSP5 — — 0.125 0.125

The BHK-T7/P5 cells were cultured in DMEM medium supplemented with 5%FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in an atmosphereof 5% CO₂ at 37° C. The medium and the cells were harvested 48 hoursafter the transfection. The harvested medium and cells were repeatedlyfreeze-thawed 3 times to prepare a cell lysate, and the cell lysate wasadded to monkey MA104 cells (ATCC CRL-2378.1) for passage. Morespecifically, about 0.5 mL of the cell lysate was added to confluentMA104 cells on 12-well plates in the presence of 0.5 μg/mL trypsin. TheMA104 cells were cultured in DMEM medium without FBS. In the case wherethe cells showed cytopathic changes during the 7 days of culture afterthe passage, artificial recombinant rotavirus production was judged assuccessful.

Results

The results are shown in Table 5.

TABLE 5 Group A Group B Group C Group D Wells with 2/24 6/24 2/24 16/24cytopathic changes/total wells

Cytopathic changes were observed in all groups, namely group A, in whichthe capping enzyme expression vectors and the FAST protein expressionvector were co-expressed with the 11 rotavirus genome segment expressionplasmids, group B, in which the capping enzyme expression vectors, theFAST protein expression vector and the NSP2 expression vector wereco-expressed with the 11 rotavirus genome segment expression plasmids,group C, in which the capping enzyme expression vectors, the FASTprotein expression vector and the NSP5 expression vector wereco-expressed with the 11 rotavirus genome segment expression plasmids,and group D, in which the capping enzyme expression vectors, the FASTprotein expression vector, the NSP2 expression vector and the NSP5expression vector were co-expressed with the 11 rotavirus genome segmentexpression plasmids. These results confirmed successful production ofartificial recombinant rotaviruses. The production efficiency was 3times higher in group B than in group A, equal between group C and groupA, and 8 times higher in group D than in group A. These results showthat the overexpression of an NSP2 gene product and/or an NSP5 geneproduct improves production efficiency.

Example 10: Improvement of Rotavirus Reverse Genetics System (2)

An experiment was performed to examine the feasibility of rotavirusproduction without using a FAST protein expression vector or cappingenzyme expression vectors.

Materials and Methods

The RNA genome segment expression vectors, the NSP2 expression vector,the NSP5 expression vector, the transfection reagent and the host cellsused in this example were the same as those in Example 9. BHK-T7/P5cells were seeded on 12-well culture plates at 4×10⁵ cells/well on theprevious day of transfection. The BHK-T7/P5 cells were transfected withthe above vectors in the combinations and DNA amounts described in Table6. The transfection reagent was used in a volume of 2 μL per microgramof DNA. The culture of the BHK-T7/P5 cells and the passage in monkeyMA104 cells were performed in the same manner as in Example 9. In thecase where the cells showed cytopathic changes during the 7 days ofculture after the passage, artificial recombinant rotavirus productionwas judged as successful.

TABLE 6 Group X Group Y DNA amount DNA amount Vector name (μg) (μg)pT7-VP1SA11 0.25 0.25 pT7-VP2SA11 0.25 0.25 pT7-VP3SA11 0.25 0.25pT7-VP4SA11 0.25 0.25 pT7-VP5SA11 0.25 0.25 pT7-VP6SA11 0.25 0.25pT7-VP7SA11 0.25 0.25 pT7-NSP1SA11 0.25 0.25 pT7-NSP2SA11 0.25 0.75pT7-NSP3SA11 0.25 0.25 pT7-NSP4SA11 0.25 0.25 pT7-NSP5SA11 0.25 0.75pCAG-NSP2 0.25 — pCAG-NSP5 0.25 —Results

In the case of overexpression of the NSP2 gene product and the NSP5 geneproduct, an artificial recombinant rotavirus was successfully producedeven without transfection of the capping enzyme expression vectors orthe FAST protein expression vector into the host cells. As a means forthe overexpression of the NSP2 gene product and the NSP5 gene product,transfection of the NSP2 expression vector and the NSP5 expressionvector in addition to the RNA genome segment expression vectors as shownin group X was proven to be useful, and also transfection of increasedDNA amounts of the expression vectors for RNA genome segments encodingNSP2 and NSP5 as shown in group Y was proven to be useful. Inparticular, the results of group Y demonstrate that an artificialrecombinant rotavirus can be produced by transfecting only the 11rotavirus RNA genome segment expression vectors into host cells andsubsequently culturing the cells.

Example 11: Production of Artificial Recombinant Attenuated VirusUtilizing Mutation in NSP4 Protein

An experiment was performed to examine the feasibility of the productionof an artificial recombinant attenuated rotavirus by introducing anartificial amino acid mutation into NSP4.

Materials and Methods

(1) Preparation of Plasmid Having an Amino Acid Mutation in NSP4 Gene

A plasmid having a mutated NSP4 gene, in which the cytosine (C) atposition 55 of the NSP4 gene (SEQ ID NO: 20) was substituted withglycine (G), was prepared from pT7-NSP4SA11 (see Example 2) as atemplate using KOD-Plus-Mutagenesis Kit (trade name, Toyobo) andspecific primers for the gene. This plasmid (designated aspT7-NSP4SA11-L5S) expresses a mutant NSP4 protein having serine (S) inplace of the leucine (L) at residue 5 of the native NSP4 protein.

(2) Production of Artificial Recombinant Virus Having a Mutation in NSP4

An artificial recombinant rotavirus having a mutation in NSP4(rsSA11/NSP4-L5S) was produced in the same manner as in Example 2 exceptthat pT7-NSP4SA11-L5S was used instead of pT7-NSP1SA11 in the set of the11 RNA genome segment expression vectors prepared in Example 2 (2). Awild-type artificial recombinant rotavirus (wild-type SA11) was alsoproduced in the same manner as in Example 2.

(3) Confirmation of Replication Capability of Artificial RecombinantRotavirus Having a Mutation in NSP4

Confluent MA104 cells on 12-well plates were infected withrsSA11/NSP4-L5S or wild-type SA11 at an MOI of 0.01. After viraladsorption at 37° C. for 1 hour, the cells were washed once with PBS andthen cultured in FBS-free DMEM supplemented with 0.5 μg/mL trypsin.After 48 hours of infection, the medium and the cells were harvested andthen freeze-thawed 3 times to prepare a cell lysate. The cell lysate wassubjected to plaque assay. The plaque assay was performed in the samemanner as in Example 1.

Results

The results are shown in FIG. 13 . The proliferation capacity ofrsSA11/NSP4-L5S was 8.7 times lower than that of wild-type SA11(21500000 vs 2450000). There has been no report on the production ofattenuated rotaviruses utilizing artificial mutation in NSP4. The NSP4mutant rotavirus produced in this example is a replication-competentattenuated virus and can be a promising vaccine candidate.

In addition, the present inventors confirmed that the rotavirus having adeletion mutation in NSP1 (rsSA11/NSP1ΔC108) produced in Example 3 and aseparately-produced rotavirus having a deletion mutation in NSP3 alsohad a lower proliferation capacity as compared with the wild-typerotavirus (data not shown). Therefore, artificial recombinantrotaviruses having an artificial mutation in NSP1 or NSP3 also arereplication-competent attenuated viruses and can be promising vaccinecandidates.

Example 12: Production of Artificial Recombinant Rotavirus StablyExpressing a Green Fluorescent Protein

An experiment was performed to examine the feasibility of the productionof a recombinant rotavirus expressing a green fluorescent protein,ZsGreen.

Materials and Methods

(1) Preparation of NSP1 Expression Plasmids Having a Green FluorescentProtein Gene Insertion

The ZsGreen (hereinafter referred to as ZsG) gene was used as the greenfluorescent protein gene. The ZsG protein-coding region (SEQ ID NO: 33)of the pZsGreen vector (Clontech) was amplified by PCR, and theamplified product was inserted between positions 111 and 112 of the NSP1gene (SEQ ID NO: 15) of pT7-NSP1SA11 to yield an NSP1 gene expressionplasmid having a ZsG gene insertion (designated aspT7-NSP1SA11-ZsG-Full). In addition, variants of plasmidpT7-NSP1SA11-ZsG-Full, namely a plasmid having a deletion of positions134 to 465 of the NSP1 gene, a plasmid having a deletion of positions134 to 855 of the same gene, and a plasmid having a deletion ofpositions 134 to 1243 of the same gene (designated aspT7-NSP1SA11-ZsG-Δ332, pT7-NSP1SA11-ZsG-Δ722 and pT7-NSP1SA11-ZsG-Δ1110,respectively), were produced (see FIG. 14 ).

(2) Production of Artificial Recombinant Viruses and Confirmation of ZsGExpression

ZsG-expressing rotaviruses were produced in the same manner as inExample 2 except that pT7-NSP1SA11-ZsG-Full, pT7-NSP1SA11-ZsG-Δ332,pT7-NSP1SA11-ZsG-Δ722 or pT7-NSP1SA11-ZsG-Δ1110 was used instead ofpT7-NSP1SA11 in the set of the 11 RNA genome segment expression vectorsprepared in Example 2 (2). The viruses produced using the differentZsG-expressing plasmids are designated as rsSA11/ZsG-Full,rsSA11/ZsG-Δ332, rsSA11/ZsG-Δ722 and rsSA11/ZsG-Δ1110. The producedviruses were separately added to infect MA104 cells, and greenfluorescence (ZsG expression) was examined under a fluorescencemicroscope.

(3) Confirmation of Retention Rate of ZsG Gene in Serial-PassagedZsG-Expressing Rotaviruses

Confluent MA104 cells on 24-well plates were infected withrsSA11/ZsG-Full, rsSA11/ZsG-Δ332, rsSA11/ZsG-Δ722 or rsSA11/ZsG-Δ1110 atan MOI of 0.0001 and cultured in FBS-free DMEM supplemented with 0.5μg/mL trypsin. Each virus strain was recovered from the culturesupernatant harvested at 72 hours postinfection and was used as stockP1. 1 μL of virus stock P1 of each strain was separately added to infectconfluent MA104 cells on 24-well plates and cultured in FBS-free DMEMsupplemented with 0.5 μg/mL trypsin for 72 hours. Then, stock P2 wasprepared. The same viral infection procedure was repeated to preparevirus stocks up to P10. Confluent MA104 cells on 12-well plates wereinfected with virus stock P1, P5 or P10 of each strain at an MOI of 0.01and cultured in DMEM without 5% FBS. After 16 hours of infection, thecells were fixed with 10% formalin for 24 hours and then subjected toimmunostaining for a viral antigen. The fixed cells were washed twicewith PBS, treated with 0.1% Triton X-100 for cell permeabilization, andreacted with a rabbit anti-rotavirus NSP4 antibody and an anti-rabbitIgG antibody-Alexa 594 conjugate for viral antigen detection. Theimmunostained cells were observed with a fluorescence microscope, andthe ZsG expression level in viral antigen-positive cells was determined.

Results

The results are shown in FIG. 15 . The ZsG expression level afterinfection with rsSA11/ZsG-Full was 100% for P1, 57.1% for P5 and 8.6%for P10, showing that ZsG expression decreased with repeated passage. Incontrast, the ZsG expression level after infection with rsSA11/ZsG-Δ332,rsSA11/ZsG-Δ722 or rsSA11/ZsG-Δ1110 ranged 99 to 100% for P1, P5 andP10, showing that the 332- to 1110-base deletion of the NSP1 gene led tostable retention of the ZsG gene.

Example 13: Improvement of Mammalian Orthoreovirus Reverse GeneticsSystem

An experiment was performed to examine whether co-expression withMammalian orthoreovirus μNS and σNS, which are functionally the same asrotavirus NSP2 and NSP5, would improve the efficiency of artificialrecombinant Mammalian orthoreovirus production.

Materials and Methods

(1) Preparation of μNS Expression Vector and σNS Expression Vector

For preparation of a NS expression vector and a σNS expression vector,the protein-coding region DNA of the μNS gene (M3 gene in Table 1,GenBank ACCESSION: AF174382, SEQ ID NO: 6) of Mammalian orthoreovirusstrain T1L and the protein-coding region DNA of the σNS gene (S3 gene inTable 1, GenBank ACCESSION: M14325, SEQ ID NO: 9) of the same strainwere individually inserted into the plasmid pCAGGS shown in FIG. 10 .These coding region DNAs were synthesized by custom gene synthesisservices (Eurofins Genomics). These synthetic DNAs were individuallyinserted into the BglII restriction site of plasmid pCAGGS (betweenpositions 1753 and 1754 of SEQ ID NO: 28) to yield pCAG-μNST1L(Mammalian orthoreovirus μNS expression vector) and pCAG-σNST1L(Mammalian orthoreovirus σNS expression vector).

(2) Production of Artificial Recombinant Virus

BHK-T7/P5 cells were seeded on 24-well culture plates at 2×10⁵cells/well on the previous day of transfection. The BHK-T7/P5 cells weretransfected with 0.4 μg each of the RNA genome segment expressionvectors produced in Example 1 (pT7-L1-M2T1L, pT7-L2-M3T1L, pT7-L3-S3T1Land pT7-S1-S2-S4-M1T1L); and 0.4 μg of the NS expression vector(pCAG-μNST1L) and/or 0.4 μg of the σNS expression vector (pCAG-σNST1L)using a transfection reagent (TransIT-LT1 (trade name), Mirus). Thetransfection reagent was used in a volume of 2 μL per microgram of DNA.The BHK-T7/P5 cells were cultured in DMEM medium supplemented with 5%FBS, 100 units/mL penicillin and 100 μg/mL streptomycin in an atmosphereof 5% CO₂ at 37° C. The medium and the cells were harvested 48 hoursafter the transfection. The harvested medium and cells were repeatedlyfreeze-thawed 3 times and used as a virus sample for plaque assay (seeExample 1), from which the viral titer was determined.

Results

As compared with the viral titer from the cells transfected with onlythe 4 expression vectors for the RNA genome segments of MRV T1L(pT7-L1-M2T1L, pT7-L2-M3T1L, pT7-L3-S3T1L and pT7-S1-S2-S4-M1T1L), theviral titer from the cells co-transfected with the μNS expression vectorand the σNS expression vector was about 8.2 times higher. The viraltiter from the cells co-transfected with only the μNS expression vectorwas about 6.4 times higher. These results show that co-transfection ofthe RNA genome segment expression vectors with the μNS expression vectoronly or with both the μNS expression vector and the σNS expressionvector into the host cells greatly improves the efficiency of artificialrecombinant virus production.

The present invention is not limited to the particular embodiments andexamples described above, and various modifications can be made withinthe scope of the appended claims. Other embodiments provided by suitablycombining technical means disclosed in separate embodiments of thepresent invention are also within the technical scope of the presentinvention. All the academic publications and patent literature cited inthe description are incorporated herein by reference.

The invention claimed is:
 1. A method for producing an artificial recombinant rotavirus, comprising: introducing a vector containing expression cassettes for 11 individual RNA genome segments of a rotavirus or introducing a set of 11 single-stranded RNA transcripts from the expression cassettes into host cells, overexpressing a rotavirus NSP2 gene product or a rotavirus NSP5 gene product in the host cells, and culturing the host cells.
 2. The method according to claim 1, wherein the rotavirus NSP5 gene product is overexpressed in the host cells.
 3. The method according to claim 1 wherein, the host cells express neither a FAST protein nor a capping enzyme.
 4. The method according to claim 1 wherein, the host cells express a FAST protein and/or a capping enzyme.
 5. The method according to claim 2 wherein, the host cells express a FAST protein and/or a capping enzyme.
 6. The method of claim 1 wherein, the host cells express a capping enzyme.
 7. The method of claim 2 wherein, the host cells express a capping enzyme.
 8. A method for producing an artificial recombinant rotavirus, comprising: introducing a vector containing expression cassettes for 11 individual RNA genome segments of a rotavirus or introducing a set of 11 single-stranded RNA transcripts from the expression cassettes into host cells, which express a FAST protein, and culturing the host cells.
 9. The method of claim 8 wherein, the host cells also express a capping enzyme.
 10. A method for producing an artificial recombinant rotavirus, comprising: introducing a vector containing expression cassettes for 11 individual RNA genome segments of a rotavirus or introducing a set of 11 single-stranded RNA transcripts from the expression cassettes into host cells, which express a capping enzyme, and culturing the host cells.
 11. The method of claim 10 wherein, the host cells also express FAST protein. 